Journ~or

Neural ~ s i o n

j. Neural Transmission 44, 21--38 (1979)

@ by Springer-Verlag 1979

d-Amphetamine-Induced Increase in Catecholamine Synthesis in the Corpus Striatum of the Rat: Persistence of the Effect After Tolerance 1 R. G. P e a r l 2 and L. S. S e l d e n 8 Department os Pharmacological and Physiological Sciences, The University of Chicago, Chicago, Illinois, U.S.A. With 5 Figures Received October 24, 1977

Summary

The effect of d-amphetamine on in vivo catecholamine synthesis in four regions of rat brain was determined by measuring the accumulation of dopa a~er inhibition of dopa decarboxylase. In doses up to 2.5 mg/kg, d-amphetamine caused dose-dependent increases in striatal dopa accumulation to a maximum of 280 ~ of control; further increases in dose resulted in smaller effects until 10 mg/kg d-amphetamine was not significantly different from control, d-Amphetamine did not alter dopa accumulation in telencephalon, in diencephalon-mesencephalon, or in pons-medulla oblongata, d-Amphetamine did not affect either dopamine levels in striatum or NE levels in pons-medulla oblongata; at high doses, d-amphetamine did reduce norepinephrine levels in telencephalon and in diencephalon-mesencephalon. Daily administration of pre-session but not of post-session d-amphetamine produced tolerance to the effects of d-amphetamine on milk consumption in rats. The ability of d-amphetamine to increase striatal catecholamine synthesis was not altered by the development of tolerance to d-amphetamine. These results suggest that tolerance to d-amphetamine is not related to its effect on catecholamine synthesis but instead occurs via This research was supported by U.S. Public Health Service National Institute of Mental Health Grant MH-01119 l- 12. Supported by Training Grant USPHS 2 TO5 GM 01939-07 (MSTP). 3 Supported by Research Career Development Award 5 KO2 MH-10562-01.

0300-9564/79/0044/0021/$ 03.60

22

R.G. Pearl and L. S. Selden:

changes in aspects of catecholamine metabolism other than synthesis via change in catecholamine release, reuptake, or receptor sensitivity, or via changes in non-catecholaminergic mechanisms. Introduction

The mechanism of action of d-amphetamine is believed to be mediated at least in part, through release of endogenous catecholamines (Stein, 1964; Weiss*nan et aI., 1966; Carlsson, 1970; Moore et al., 1970; Scheel-Krigger, 1971; Baez, 1974). The ability of damphetamine to augment the in vivo release of dopamine from striatum has been extensively documented (Glowinski et al., 1966; McKenzie and Szerb, 1968; Besson et al., 1971; Chiueh and Moore, 1975). Despite this increased release, dopamine levels after low or moderate doses of d-amphetamine remain unchanged or increase (Leonard and Shallice, 1971; Riffee and Gerald, 1976; Javoy et al., 1974; Koda and Gibb, 1973; Harris et al., 1975; Kogan et al., 1976). This finding suggests that striatal dopamine synthesis increases following d-amphetamine administration. The majority of in vivo measurements of dopamine synthesis atter d-amphetamine administration do demonstrate such an effect (Costa and Groppetti, 1970; Costa et aI., 1972; Roth et aI., 1974; Kehr et al., 1977; Heffner et al., 1977). The accumulation of dopa aflcer inhibition of aromatic amino acid decarboxylase with NSD 1015 (3-hydroxybenzylhydrazine) has been shown to measure the in vivo rate of catecholamine synthesis (Carlsson et al., 1972). We have used this technique to measure catecholamine synthesis in striatum and in three other brain regions. Since the majority of studies of the effect of d-amphetamine on striatal dopamine synthesis either have used only one dose of drug or have used only a few subjects for each data point, we have investigated the entire dose-response range of 0.625 mg/kg to 10 mg/kg d-amphetamine with at least 15 subjects per data point. Tolerance, a decreased effectiveness of a drug atter repeated administration of the drug, occurs to many of the effects of amphetamine (Schuster and Zirnmerrnan, 1961; Tilson and Sparber, 1973; ChieI et al., 1974; Magour et al., 1974; Mac Phail and Selden, 1976; Pearl and Selden, 1976). One factor in the development of tolerance to d-amphetamine may be a chronic change in the degree of activation of catecholaminergic systems and/or a change in the ability of d-amphetamine to activate these systems (Goldstein et aI., 1974). In general, experiments which have investigated such changes either have found no changes (Lewander, 1971; Schanberg and Cook, 1972;

d-Amphetamine-Induced Increase in Catecholamine Synthesis

23

Sparber and Tilson, 1972; Hulme et al., 1974) or have been unable to convincingly correlate such changes to the development of tolerance (Fibiger and McGeer, 1971; Koda and Gibb, 1973). Since changes in the activation of catecholaminergic systems can affect the rate of catecholamine synthesis (Weiner, 1970; Carlsson et al., 1974; Costa et al., 1974; Roth et al., 1974), the rate of catecholamine synthesis before and ai%r d-amphetamine pretreatment in tolerant and nontolerant rats should indicate if such changes have occurred. Since the acute administration of d-amphetamine altered catecholamine synthesis only in striatum, we investigated the effects of tolerance to d-amphetamine on striatal catecholamine synthesis both with and without acute pretreatment with d-amphetamine. Carlton and Wolgin (1971) and Campbell and Selden (1973) have shown that the repeated administration of d-amphetamine is not, in itself, sufficient to cause tolerance to the disruptive effects of damphetamine on milk consumption and on differential-reinforcementof-low-rate (DRL) 17.5-sec behavior, respectively. In both experiments, tolerance developed only if the drug was administered prior to the session so that it altered the behavior; repeated post-session administration of d-amphetamine did not result in tolerance. Rats receiving daily post-session d-amphetamine can therefore serve as a control for separating the biochemical effects of repeated administration of d-amphetamine from those of tolerance. Accordingly, we have compared daily post-session d-amphetamine with daily presession d-amphetamine for the effects on striatal catecholamine synthesis. Methods

Sub)ects and Apparatus Male Sprague-Dawley rats (Holtzman Co., Madison, WI) initially 60 days old were used in both experiments. In Experiment I, 102 rats were housed two per cage in a room maintained at 24 ~ and illuminated 12 hours per day. In Experiment II, 60 rats were housed individually. Water was available ad Iibitum in Experiments I and II; food ('Purina rat chow) was available ad libiturn in Experiment I and limited as described in Experiment II.

Experiment I: Effect of Single Doses of d-Amphetamine Sulfate on Catecholamine Synthesis in Rat Brain Rats received one of six doses (0, 0.625, 1.25, 2.5, 5, 10mg/kg) of d-amphetamine sulfate 10min aider receiving 50mg/kg NSD 1015. Rats were killed by decapitation one hour aider receiving NSD 1015 (50 min

24

R.G. Pearl and L. S. Seiden:

afer receiving d-amphetamine sulfate). Brains were rapidly removed from the skull and dissected on an ice-cold plate by a modification of the method of Glowinski and Iversen (1966). The following sections were used: 1. Striatum (caudate nucleus, globus pallidus and putamen), 2. Telencephalon (cortex, hippocampus, amygdala), 3. Diencephalon-mesencephalon, 4. Ports-medulla oblongata. Briefly, a coronal cut was made anterior to the olfactory tubercle; the anterior portion was saved as part of the telencephalon. A second cut was made at the posterior border of the optic chiasm, and the resulting piece was turned on its rostral surface. Two portions were cut from this piece, each using the corpus callosum as a dorsal boundary, the lateral ventricle as a medial boundary, and the amygdala-globus pallidus junction as a ventral boundary. All grossly myelinated fiber was removed from each of the 2 pieces which were then combined to give the caudate sample. A shallow sagittal cut was made along the dorsal surface of the brain and the remaining left and right telencephalic tissue was peeled. These pieces were combined with the tissue anterior to the olfactory tubercle to give the telencephalon sample. A transverse cut Was made from just posterior to the mamillary bodies to just posterior to the inferior colliculi. The resulting piec e was termed diencephalon-mesencephalon. The cerebellum was removed from the remaining piece which then constituted the pons-medulla oblongata section. Samples were frozen in liquid nitrogen until assayed.

Experiment II: Effect of Single and Repeated Administration of d-Amphetamine on CatechoIamine Synthesis in Striatum of Rat Brain A milk consumption procedure modified from Carlton and Wolgin (1971) was used. Sixty rats received 30-minute periods of access daily to a milk solution ('one part sweetened condensed milk, two parts water) 20 rain atter being weighed and injected with either saline or d-amphetamine (2.5 mg/kg). Twenty minutes afer each session, rats were injected with either saline or d-amphetamine (2.5 mg/kg) and given enough food pellets to maintain body weight at 300 g. For 10 days saline was administered before and after each session. For the next 36 days (with the exception of day 27) 22 rats received pre-session d-amphetamine (2.5 mg/kg) and post-session saline (A-S group), 15 rats received pre-session saline and post-session d-amphetamine (2.5 mg/kg) (S-A group), and 23 rats continued to receive pre-session saline and postsession saline (S-S group). In order to test for tolerance, all rats received pre-session d-amphetamine and post-session saline on day 27 of drug administration. On the final day of the experiment (day 37), all rats received 50 mg/kg NSD 1015 (3-hydroxyhenzylhydrazine HC1) 30 rain prior to the session. Rats received either saline or d-amphetamine 10 rain later (20 min prior to the session). In order to assess the effect of milk consumption on catecholamine turnover, some animals were not allowed access to milk during this

d-Amphetamine-Induced Increase in Catecholamine Synthesis

25

final session. The 8 treatment groups used are described more fully in Results. All animals were killed by decapitation at the end of the session; brains were rapidly removed from the skull and dissected to give the striatum section as described for Experiment I.

Catecholamine Assay Dopa and either doparnine (striatum) or norepinephrine (telencephalon; diencephalon-mesencephalon; pons-medulla oblongata) were extracted from single brain parts according to Carlsson et al. (1972) by means of a strong cation exchange column (Dowex 50W-X4). Dopa was oxidized to a fluorescent indole and determined by the method of Kehr et al. (1972 a). Dopamine was determined by the method of Carlsson and Lindqvist (1962) as modified by Schoenfeld and Selden (1969). Norepinephrine was determined by the method of Bertler et al. (1958). Recovery was 68 % for dopa, 69 % for dopamine and 65 ~ for norepinephrine.

Analysis of Data Dopa, dopamine and norepinephrine concentrations are expressed as #g/g wet tissue; values are corrected for recovery. In Experiment 1, a oneway analysis of variance using the six doses of d-amphetamine was performed for each compound and each brain part (Winer, 1962); comparisons among treatment means were made by the Newman-Keuls test whenever the analysis of variance was significant. In Experiment II, a one-way analysis of variance using the 8 treatment groups was performed for dopa and for dopamine concentrations in the striatum; the significance of different treatments was then tested by means of orthogonal comparisons (Winer, 1962). In Experiment II, milk bottles were weighed before and atter each session and the difference was recorded to the nearest gram. Non-parametric statistics were used to compare the milk consumption of different groups because the assumption of homogeneity of variance required for parametric tests (Winer, 1962) was frequently violated. For day 27, differences in milk consumption among the 3 daily treatment groups were assessed by the Kruskal-Wallis one-way analysis of variance with subsequent MannWhitney U-tests (Siegel, 1956). For the final day treatment, the effect of NSD 1015 administration was assessed by means of Wilcoxon matched-pairs signed-ranks tests and Mann-Whitney U-tests as described in Results (Siegel, 1956).

Chemicals d-Amphetamine sulfate was obtained from the Smith Kline and French Laboratories (Philadelphia, PA). NSD 1015 (3-hydroxybenzylhydrazine HC1) was obtained from Dr. Per Martinson (The University of G6teborg, Sweden). All drugs were dissolved in 0.9 % saline and injected intraperitoneally in a volume of 1 ml/kg. All doses are expressed in terms of the respective salts.

26

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d-Amphetamine-Induced Increase in Catecholarnine Synthesis

27

Results

Experiment I: Effect of Single Doses of d-Amphetamine Sulfate on Catecholamine Synthesis in Rat Brain The dose-response curves for the effect of d-amphetamine on dopa accumulation in each of 4 rat brain regions a~er dopa decarboxylase inhibition with NSD 1015 are presented in Fig. 1, the corresponding levels of DA (striatum) and NE (remaining 3 brain regions) are presented in Fig. 2. As reported by Kehr et al. (1977), d-amphetamine 0 I.Z

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Fig. 2. Dose-response curves for the effect of d-amphetamine on dopamine (DA) levels in striatum and on norepinephrine (NE) levels in telencephalon, in diencephalon-mesencephalon, and in pons-medulla oblongata of rat brain. Rats received 50 mg/kg NSD 1015 one hour before sacrifice and either saline or d-amphetamine 50 rain before sacrifice. Brackets represent _-4_-I standard error. N for each group was 15--17. Control values (corrected for recovery) were 10.5fig DA/g caudate, 0.42fig NE/g telencephalon, 1.03fig NE/g diencephalon-mesencephalon, and 0.73/~g NE/g pons-medulla oblongata. * Less than saline control (p < .05; p < .01) for 10 mg/kg dose and 0.625 mg/kg dose (p < .05). + Less than saline control (p < .05), 0.625 mg/kg dose (p